Oilseed from Brassica plants is an increasingly important crop. As a source of vegetable oil, it presently ranks behind only soybeans and palm in commercial importance and it is comparable with sunflowers. The oil is used both as a salad oil and as a cooking oil.
In its original form, Brassica oil, known as rapeseed oil, was harmful to humans due to its relatively high level of erucic acid, Erucic acid is commonly present in native cultivars in concentrations of 30 to 50 percent by weight based upon the total fatty acid content. This problem was overcome when plant scientists identified a germplasm source of low erucic acid rapeseed oil (Stefansson, 1983).
In addition, plant scientists have attempted to improve the fatty acid profile for rapeseed oil (Robbelen, 1984; Ratledge et al., 1984; Robbelen et al., 1975; and Rakow et al., 1973). These references are representative of those attempts.
Particularly attractive to plant scientists were so-called xe2x80x9cdouble-lowxe2x80x9d varieties: those low in erucic acid in the oil and low in glucosinolates in the solid meal remaining after oil extraction (i.e., an erucic acid content of less than 2 percent by weight based upon the total fatty acid content, and a glucosinolate content of less than 30 xcexcmol/gram of the oil-free meal). These higher quality forms of rape, first developed in Canada, are known as canola.
More recently, plant scientists have focused their efforts on reducing the glucosinolate content further, to levels of less than 20 xcexcmol/gram of oil-free meal, as verified by quantifying trimethylsilyl (TMS) derivatives (Sosulski and Dabrowski, 1984) for spring canola, or less than 20 xcexcmol/gram of whole, ground seed, as determined by high performance liquid chromatography (HPLC) (International Organization for Standardization, reference number ISO 9167-30 1:1992(E)) for winter canola.
Glucosinolates are sulfur-based compounds that remain in the solid component of the seedxe2x80x94the solid mealxe2x80x94after the seed has been ground and its oil has been extracted. Their structure includes glucose in combination with aliphatic hydrocarbons (3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3-butenyl glucosinolate, and 2-hydroxy-4-pentenyl glucosinolate) or aromatic hydrocarbons (3-indoylmethyl glucosinolate, 1-methoxy-3-indoyl methyl glucosinolate). Aliphaitic glucosinolates are also known as alkenyl glucosinolates. Aromatic glucosinolates are also known as indoles.
High levels of glucosinolates are undesirable because they produce toxic by-products when acted upon by the enzyme myrosinase. Myrosinase is a naturally occurring enzyme present in Brassica species. When Brassica seed is crushed, myrosinase is released and catalyzes the breakdown of glucosinolates to produce glucose, thiocyanates, isothiocyanate and nitriles. When separated from glucose, these other products are toxic to certain mammals. Isothiocyanate, for example, inhibits synthesis of thryroxine by the thyroid and has other anti-metabolic effects (Paul et al., 1986). Attempts have been made to inactivate the enzyme myrosinase (using steam, for example). These attempts have not been entirely successful.
Rapeseed possesses high levels of glucosinolates (from 100 xcexcmol/gram to 200 xcexcmol/gram of oil-free meal), whereas canola possesses lower levels of glucosinolates (less than 30 xcexcmol/gram of oil-free meal). The levels of glucosinolates in canola are regulated in many countries. In Europe, for example, winter canola must have a glucosinolate content of less than 25 xcexcmol/gram of seed at 8.5% moisture, as measured by HPLC. In Canada, spring canola must have a glucosinolate content of less than 30 xcexcmol/gram of oil-free meal at 0% moisture, as measured by TMS. Many countries are requiring even lower levels of glucosinolates in order to register canola varieties.
In developing improved new Brassica varieties, breeders use self-incompatible (SI), cytoplasmic male sterile (CMS) and nuclear male sterile (NMS) Brassica plants as the female parent. In using these plants, breeders are attempting to improve the efficiency of seed production and the quality of the F1 hybrids and to reduce the breeding costs. When hybridisation is conducted without using SI, CMS or NMS plants, it is more difficult to obtain and isolate the desired traits in the progeny (F1 generation) because the parents are capable of undergoing both cross-pollination and self-pollination. If one of the parents is a SI, CMS or NMS plant that is incapable of producing pollen, only cross pollination will occur. By eliminating the pollen of one parental variety in a cross, a plant breeder is assured of obtaining hybrid seed of uniform quality, provided that the parents are of uniform quality and the breeder conducts a single cross.
In one instance, production of F1 hybrids includes crossing a CMS Brassica female parent, with a pollen producing male Brassica parent. To reproduce effectively, however, the male parent of the F1 hybrid must have a fertility restorer gene (Rf gene). The presence of a Rf gene means that the F1 generation will not be completely or partially sterile, so that either self-pollination or cross pollination may occur. Self pollination of the F1 generation to produce several subsequent generations is important to ensure that a desired trait is heritable and stable and that a new variety has been isolated.
One Brassica plant which is cytoplasmic male sterile and is used in breeding is ogura (OGU) cytoplasmic male sterile (R. Pellan-Delourme et al., 1987). A fertility restorer for ogura cytoplasmic male sterile plants has been transferred from Raphanus sativus (radish) to Brassica by Institut National de Recherche Agricole (INRA) in Rennes, France (Pelletier et al., 1987). The restorer gene, Rf1 originating from radish, is described in WO 92/05251 and in Delourme et al., (1991).
However, this restorer is inadequate in that restorer inbreds and hybrids carrying this Rf gene have elevated glucosinolate levels and the restorer is closely related to a decrease in seed setxe2x80x94the number of ovules per siliquexe2x80x94(Pellan-Delourme et al., 1988; Delourme et al., 1994). In the case of hybrids, the glucosinolate levels are elevated even when the female parent has reduced glucosinolate content. These levels, typically more than 30 xcexcmol/gram of oil-free meal, exceed the levels of glucosinolates allowable for seed registration by most regulatory authorities in the world. Thus, the restorer can be used for research purposes, but not to develop directly canola-quality commercial hybrid varieties. To date, there is no other source of a restorer of fertility for ogura cytoplasmic male sterility available.
INRA outlines the difficulties associated with obtaining restorer lines with low glucosinolate levels for ogura cytoplasmic sterility (Delourme, et al., 1994; Delourme, et al., 1995). INRA indicates that these difficulties are due to the linkage between male fertility restoration and glucosinolate content in its breeding material. INRA suggests that more radish genetic information needs to be eliminated in its restorer lines (Delourme, et al., (1995)). Although improvements have been made to restorers during the past few years, isozyme studies performed on the improved restorer lines indicate that radish genetic information still remains around the restorer gene (Delourme et al., 1994).
INRA has attempted to develop a restorer having decreased glucosinolate levels. It reported a heterozygous restorer with about 15 xcexcmol per gram (Delourme et al., 1995). However, (i) this restorer was heterozygous (Rfrf) not homozygous (RfRf) for the restorer gene, (ii) this restorer was a single hybrid plant rather than an inbred line, (iii) there was only a single data point suggesting that this restorer had a low glucosinolate level rather than multiple data points to support a low glucosinolate level, (iv) there was no data to demonstrate whether the low glucosinolate trait was passed on to the progeny of the restorer, and (v) the restorer was selected and evaluated in a single environmentxe2x80x94the low glucosinolate trait was not demonstrated to be stable in successive generations in field trials. INRA has not introduced commercially any homozygous restorer having low glucosinolate levels. Its restorer (reported in Delourme et al., 1995) cannot be used to develop restorer inbreds or single cross hybrids products (where the restorer is used as a male inbred) with decreased glucosinolate levels for commercial development.
Canadian patent application 2,143,781 of Yamashita, et al., published on Sep. 11, 1995, claims a hybrid breeding method for crop plants in the family Brassicaceac in which an F1 seed is produced by crossing the female parent of a self-incompatible male sterile line with a male parent. In one embodiment, the male parent possesses a fertility restorer gene. The fertility restorer gene (IM-B) is for MS-N1-derived cytoplasm and was derived from a winter variety (IM line). This was then crossed with a spring double-low line (62We). Although this restorer is alleged to result in low glucosinolate levels, it is not a restorer for ogura cytoplasmic male sterility.
Other breeders have attempted to introduce Rf genes from radish into rapeseed plants by means of intergeneric crossing. However, these crosses have not been employed practically. Canadian patent application 2,108,230 of Sakai, et al, published on Oct. 12, 1993, claims a fertility restorer gene of a Raphanus plant which is introduced into a Brassica plant by cell fusion or intergeneric cross. This application does not disclose (1) a restorer of ogura cytoplasmic male sterility which maintains decreased glucosinolate levels in the oilseed of an F1 generation or (2) the advantageous use of a restorer to develop restorer inbreds and to develop single cross hybrid combinations for commercial products (where the restorer is used as a male inbred).
To attempt to avoid the high glucosinolate content of INRA""s restorer of ogura cytoplasmic male sterility, INRA and Serasem (UNCAC) have developed a Brassica napus variety called SYNERGY(copyright). SYNERGY is a cross of ogura cytoplasmic male sterile SAMOURAI(copyright) (bred by INRA) and male fertile FALCON(copyright) (bred by NPZ). FALCON does not carry the restorer gene for ogura cytoplasmic male sterility. Therefore, the F1 hybrid is male sterile. SYNERGY is sold as a xe2x80x9ccomposite hybrid linexe2x80x9d (CHL) which consists of a blend of roughly 80% male sterile F1 hybrid (SYNERGY) and 20% male fertile (FALCON), which provides pollen for seed-set on the male sterile F1 plants in the farmer""s field.
There are a number of difficulties, however, in relying upon a composite hybrid line. The most important are: (1) that Brassica napus is a self-pollinating species, so under poor pollination conditions (such as prolonged cool, wet weather) there may be inadequate pollen movement from the male fertile plants to the F1 hybrid plants, resulting in poor seed set and yield, and (2) that the F1 hybrid plants are more vigorous than the FALCON plants, so the former may outcompete the latter, resulting in too little pollen being available for optimal seed set and yield on the F1 plants.
To date, no one has been able to develop an improved restorer having a homozygous (fixed) restorer gene (RfRf) for ogura cytoplasmic male sterility whose oilseeds have low glucosinolate levels. The restorer must be homozygous (RfRf) so that it can be used to develop restorer inbreds or, as male inbreds, in making single cross hybrid combinations for commercial product development. Ideally, glucosinolate levels would be well below those set out in standards for canola in various countries. That way, breeders could use the improved restorer to produce Brassica inbreds and hybrids having oilseeds with low glucosinolate levels. This would benefit farmers, who could then plant Brassica hybrids which, following pollination, would yield oilseeds having low glucosinolate levels and other desirable characteristics.
In many counties, oilseeds produced by farmers for crushing or for export are not checked for their glucosinolate content. Sometimes a particular lot of canola may have high glucosinolate content, resulting in contamination of the bulk grain to which the poor quality canola is added. It would be an improvement if the glucosinolate content of oilseeds was well below the standards set by various countries in order to avoid contamination of the bulk grain.
Thus, there remains a need for an improved Brassica plant which is a homozygous restorer of fertility for ogura cytoplasmic male sterility and which produces an oilseed with low glucosinolate content. To date, Brassica plants which are restorers of fertility for ogura cytoplasmic male sterility (i) have been heterozygous, rather than homozygous (fixed), for the restorer trait, or (ii) have not produced oilseeds with low glucosinolate content. Indeed, glucosinolate content of such oilseeds has been higher than 30 xcexcmol/gram of oil-free meal.
It is an object of the present invention to provide an improved mature Brassica plant which is a homozygous restorer for ogura cytoplasmic male sterility and which has a glucosinolate content of less than 30 xcexcmol/gram of seed. This restorer could be used to produce restorer inbreds or hybrids with low glucosinolate content. This would allow production of fully-restored, single cross hybrids with genetically-low glucosinolate content in both the hybrid seed and in the oilseed harvested from the hybrid plants.
It is an object of the present invention to provide a Brassica oilseed of the Brassica plant containing a nuclear restorer for ogura cytoplasmic male sterility and having an improved glucosinolate level.
It is another object of the present invention to provide improved Brassica inbred lines, using the restorer. Another object is to use the restorer as a male inbred in making single cross hybrid combinations to develop commercial products.
It is another object of the present invention to provide an oil and edible vegetable meal having an improved glucosinolate level following simple crushing and extraction.
These and other objects and advantages of the invention will be apparent to those skilled in the art from a reading of the following description and appended claims.
This invention relates to a Brassica plant comprising a homozygous fertility restorer gene for ogura cytoplasmic male sterility, wherein upon pollination the plant yields oilseeds having a total glucosinolate content of not more than 30 xcexcmol per gram, 25 xcexcmol per gram or 20 xcexcmol per gram.
The oilseed of a Brassica plant comprising a homozygous fertility restorer gene for ogura cytoplasmic male sterility and having a glucosinolate content of less than than 30 xcexcmol per gram, 25 xcexcmol per gram or 20 xcexcmol per gram, may be used for preparing oil and/or meal.
This invention also relates to a Brassica plant comprising a homozygous fertility restorer gene for ogura cytoplasmic male sterility, wherein upon pollination the plant yields oilseeds having (i) a total glucosinolate content of not more than 30 xcexcmol per gram and an erucic acid content of no more than 2 percent by weight based upon the total fatty acid content, (ii) a total glucosinolate content of not more than 25 xcexcmol per gram and an erucic acid content of no more than 2 percent by weight based upon the total fatty acid content or (iii) a total glucosinolate content of not more than 20 xcexcmol per gram and an erucic acid content of no more than 2 percent by weight based upon the total fatty acid content.
The Brassica plant may be Brassica napus, Brassica campestris or Brassica juncea. It may be designated as 95SN-9369, 96FNW-1792, 96FNW-1822, 96FNW-1348, 96FNW-1628 or their sub-lines. The sub-lines may be selected from a group consisting of 97SN-1650 (sub-line of 95SN-9369), 97SN-1651 (sub-line of 95SN-9369), 96FNW1792-03 (sub-line of 96FNW-1792), 96FNW1822-07 (sub-line of 96FNW1822) and 96FNW1822-08 (sub-line of 96FNW1822).
An inbred Brassica plant may be produced using this plant. A hybrid Brassica plant may be produced using this plant. Upon pollination, the inbred or hybrid plant yields oilseed having a total glucosinolate content of (i) not more than 30 xcexcmol per gram, (ii) not more than 25 xcexcmol per gram, or (iii) not more than 20 xcexcmol per gram.
This invention also includes an oilseed of the Brassica plant or from the inbred or hybrid Brassica plant. The oilseed may be present as a component of a substantially homogeneous assemblage of oilseeds which possess the specified glucosinolate content. Oil of the oilseed is also part of this invention. The oilseed may be formed on Brassica napus, Brassica campestris or Brassica juncea. The mature Brassica oilseed is capable of yielding an endogenous vegetable oil having a glucosinolate content of no more than (i) 30 xcexcmol per gram, (ii) 25 xcexcmol per gram, or (iii) 20 xcexcmol per gram.
Meal which is substantially oil free and which is produced from this oilseed is also part of this invention. The meal has a glucosinolate content of no more than (i) 30 xcexcmol per gram, (ii) 25 xcexcmol per gram, or (iii) 20 xcexcmol per gram.
This invention also relates to a part of the Brassica plant of this invention. The plant part may be selected from a group consisting of nucleic acid sequences (RNA, mRNA, DNA, cDNA), tissue, cells, pollen, ovules, roots, leaves, oilseeds, microspores, vegetative parts, whether mature or embryonic.
The Brassica plant of this invention may be used to breed a novel Brassica line. The breeding may be selected from a group consisting of isolation and transformation, conventional breeding, pedigree breeding, crossing, self-pollination, haploidy, single seed descent and backcrossing.